This invention relates to a method and apparatus for producing a cell suspension layer of a predetermined cell density (or cell spacing) on a substrate, and more particularly, to a system which senses the cell density on the substrate and automatically terminates the thickness reduction when a predetermined cell density is achieved.
In many areas of medical and biological work cell monolayers are visually examined to derive diagnostic information about the patient. Common examples of this are the examination of the blood film in the differential blood cell count and the examination of cervical cells in the Pap test. The classical manual methods for preparing cell monolayers for examination unfortunately produce cell densities of greatly varying thicknesses. Recently a method has been develloped which allows a very uniform monolayer of cells to be produced from a suspension of cells in a liquid.
U.S. Pat. Nos. 3,577,267 by Preston et al and 3,705,048 by Staunton describe two apparati which implement a method that is generally known as "spinning," in which a microscope slide or other carrier substrate is wetted with an excess of the cell suspension and spun rapidly. The excess cell suspension is quickly spun off by centrifugal force and a uniform monolayer forms on the substrate. When the monolayer has achieved the proper cell density, spinning is stopped. Because of the variations of properties of naturally occurring biological cell suspensions, the optimum spin time to produce a monolayer with ideal cell separation is not constant, but instead varies from sample to sample. In the two abovementioned patents, it is necessary to determine the optimum spin time by trial and error for each sample. The empirical determination of optimum spin times greatly reduce the usefulness of the method for high volume tests.
In U.S. Pat. No. 3,827,805, Mansfield et al, there is described a control system for a blood film spinner which overcomes the necessity for the trial and error determination of the optimum spin time. In this apparatus a beam of light is focused through the transparent substrate and the cell suspension during the spinning operation. The reduction in the transmitted light caused by the absorption or scattering in the cell suspension is measured by a direct light detector, and the amount of scattered light is summed by a large scattered light detector. The ratio of the outputs of the two detectors is compared to an exponentially decaying signal. The spinning is terminated when a predetermined relationship exists between the ratio signal and the exponentially decaying signal.
The device described by Mansfield in the aforementioned patent has several apparent shortcomings. The use of direct light as a control signal introduces inaccuracies because a reduction in direct light transmission may be caused by factors other than cell suspension thickness or cell spacing. Although the total amount of scattered light is a measure of the thickness of the cell monolayer, it can be influenced by other factors, such as, changes in the refractive index of the blood plasma relative to the refractive index of the cells. Thus, the ratio of transmitted-to-scattered light can vary for each cell spacing with the concomitant possibility of an erroneous spin time.
Similarly, the summing of all scattered light in a single scattered light sensor reduces the sensitivity of the measurement which can be achieved if the relative intensities of different parts of the pattern of scattered light are measured individually. The summing of light from all parts of the scattered light pattern can mask large changes in the proportion of light being scattered into different parts of the pattern while the total amount of scattered light remains relatively constant. This can lead to a lack of sensitivity and errors in a Mansfield device.